Evaluation of Bioinseticide in the Control of Plutella xylostella (Linnaeus, 1758): A Laboratory Study for Large-Scale Implementation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents the interesting dual purpose plant protection aptitude of the extraction from Simarouba sp.. Indeed, the notable properties of Simarouba sp. in crop protection have been pointed out by several independent research groups, Especially its chemical composition has been identified very meticulously. The experimental work has been quite well organized, but the writing style is not adequate and an minor revision is necessary before the manuscript can be accepted for publication.

Quassinoids are important active ingredients in Simarouba sp and have been proven to have insecticidal activity against pests such as aphids, as well as resistance to plant viruses. The author should focus on comparing the effects of such substances on insects in the discussion.

Author Response

Comments and Suggestions for Authors

Round 1 – Reviewer 1

Manuscript ID: sustainability-3414907

 

Title: Evaluation of Bioinsecticides in the Control of Plutella xylostella (Linnaeus, 1758): A Laboratory Study for Large-Scale Implementation

 

We appreciate the reviewer's valuable suggestions. We inform that we understood the suggestions and some sentences have been altered as indicated by this reviewer and other reviewers. Language and textual fluency have also been improved. Scientific names were reviewed.

 

Reviewer: This manuscript presents the interesting dual purpose plant protection aptitude of the extraction from Simarouba sp.. Indeed, the notable properties of Simarouba sp. in crop protection have been pointed out by several independent research groups, Especially its chemical composition has been identified very meticulously. The experimental work has been quite well organized, but the writing style is not adequate and an minor revision is necessary before the manuscript can be accepted for publication.

Quassinoids are important active ingredients in Simarouba sp and have been proven to have insecticidal activity against pests such as aphids, as well as resistance to plant viruses. The author should focus on comparing the effects of such substances on insects in the discussion.

 

Authors: Thank you for your comment. The review on quassinoids and their effects on insects has been added to the discussion (line 467).

 

Quassinoids can be classified as taxonomic markers of Simaroubaceae, as they are synthesized almost exclusively by members of this family [23, 24]. Previous studies have indicated that quassinoids can inhibit feeding through activating systems that identify substances with a bitter taste [72-74]; this finding was confirmed in the present study. A bitter taste is commonly associated with toxic substances that cause aversive responses in insects [69]. Thus, the recognition of bitter compounds is essential ability for insects, mainly due to the diversity and toxicity of these compounds [69,75].

Different quassinoids have been identified as insect-feeding and growth regulator agents in the literature [76]. Previous studies have shown insecticidal activities of quassinoids against Tetranychus urticae, Myzus persicae, Meloidogyne incognita [77], and Rhodnius milesi [78]. Further, they also showed anti-feeding potential against Locusta migratoria migratorioides [79] and other agricultural pests [80].  Specifically, quassin, simalikalactone D, bruceantin, glaucarubinone, and isobrucein demonstrated significant feeding deterrence against the Mexican bean beetle (Epilachna varivestis) and the Diamondback moth (Plutella xylostella) [81].

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

General comments

This MS describes the effect of aqueus extracts of Simarouba sp. leaves on oviposition, feeding and larval hatching of Plutella xylostella. This MS should be improved with concern to the English quality. In addition, the authors should determine and mention the Simarouba species. The title needs improvement to specify that some aqueous leaf extracts were studied and not some bioinsecticides. In addition, this MS would gain added value from the explicit determination of the chemical ingredients of the leaf extracts.

 

Specific comments

Abstract

Abstract, lines 29-32. “The present study analyzed the effects of bioinsecticides of Simarouba sp. at concentrations of 0.1, 1, 5, and 10% and a control on the feeding preference, oviposition and embryonic development of P. xylostella.”

The authors studied the effect of aqueous extracts of Simarouba sp. Leaf and not of some bioinsecticides. Please improve.

 

Abstract, lines 32 to 33. “ The results showed that the bioinsecticides of Simarouba sp. decreased oviposition and feeding in P. xylostella, and a reduction in larval hatching was also observed, indicating ovicidal properties. Please improve with respect to grammar and syntax.

 

Abstract, line 35. “Dietary concentrations of 10% and 5% caused…”. Please explain the meaning of dietary concentrations.

 

Abstract, lines 37-41.  “This study highlights the efficacy of bioinsecticides of Simarouba sp. in the control of the crucifer moth, as larval feeding and the number of in-38 dividuals reaching the larval stage, the stage at which the insect causes losses to producers, were reduced. Thus, this laboratory study is expected to be successful if practiced on 40 a large scale.” Please improve with respect to grammar and syntax.

 

Material and methods

P. 97. The P. xylostella rearing methodology was adapted from Barros et al. [25]. The authors should briefly describe the rearing methodology.

 

Line 108 to 109. After collection, the leaves were washed under running water, dried in a forced-air oven for 72 hours at 45 °C and grounded in an industrial knife mill until a fine powder was obtained. Drying the leaves at 45°C may cause substantial changes in the chemical composition. Please comment on it. In addition, give some information about the size of the leaf particles after grounding and clarify whether sieves were used.

 

Lines 112 to 115.  The authors mention that “ ..The maceration technique was used to obtain the bioinsecticides (extract aqueous) of Simarouba sp. (EA-S). Bioinsecticides with concentrations of 10, 5, 1 and 0.1% were obtained by adding 3 g of the powdered material to 30 ml of distilled water, 1.5 g to 30 ml, 0.3 g to 30 ml and 0.015 g to 30 ml. The powdered material was weighed on an analytical balance (Bel Mark Analytical Balance – 0.001 g). The solutions were left to stand for 24 hours in a refrigerator and were subsequently filtered using filter paper”.

These concentrations refer to the leaf powder and not to a bioinsecticide. Please improve.

 

Line 130- 131. The authors mention that “...the leaf area consumed was measured using ImageJ software, and the food preference index (FPI) of Kogan and Goeden [26] was calculated...”  Please give a more detailed description of the method for determining food preference.

 

Lines 141-142. The authors mention that ‘’’Subsequently, 4-cm Ø cabbage discs were immersed in EA-S at concentrations of 0.1, 1, 5 and 10%, and control discs were immersed in distilled water. Please explain the EA-S meaning. In addition, substitute “Subsequently” with “For the experiments…” Please improve.

 

Lines 148-150. The authors mention that “…A moistened filter paper disc measuring 8 cm Ø and four cabbage discs measuring 4 cm Ø (two control discs (immersed in water) and two discs immersed in the EA-S) were added to each cage as oviposition substrates…. “ . If the filter paper is 8 cm in diameter and the leaf discs 4 cm, then the leaf discs are expected to attach each other. However, Fig.2, shows a distance between the leaf discs. Please clarify.

 

Statistical analysis

Lines 177- 178. “...The experiment was conducted with a completely randomized design with 10 replicates and 3 subsamples…”. Please explain the meaning of sub-samples.

Line 179. The authors mention that the “…The data were subjected to analysis of variance (ANOVA) with a Gaussian distribution, and the means were compared using the t test at a 5% probability...”. Please explain which means were compared. 

Lines 183 -185. The authors mention that”…The effect of the plant extract was evaluated using the FPI [26]. The extract was classified as a phagostimulant if the index was greater than 1, neutral if it was equal to 1, and 184 phagodeterrent if it was lower than 1, using the formula FPI = 2A/(M+A), where A is the 185 consumed area of the treated discs and M is the consumed area of the untreated discs”. This part does not present any statistical analysis and, therefore, should be transferred to the “Materials and Methods”. The same for the text in lines 197 to 202.

Line 212. The authors mention that “The extracts were lyophilized (Alpha 1–2 LD Plus) …” . They should add some information about the extracts.

 

Results

In Table 2, it is shown that the number eggs laid in the control discs varies from 60.4 to146.0. The authors should discuss this high variability in egg production, of the control females, between the different treatments

In Table 3, the mean number (± SE) of hatched P. xylostella larvae from eggs exposed to different concentrations of Simarouba sp. aqueous extract is shown. However, the number of eggs tested is not shown and therefore, it is risky to make any comparisons. Please add information about the initial number of eggs tested and hatchability percentages.

The authors determined the quantities of tannins, phenolic compounds, and flavonoids of Simarouba sp. leaves extracts (Table 4). However, they have not determined the chemical compounds of the leaf extracts which could be of great importance.

 

Discussion

Lines 344 -346. “Our results showed that the aqueous extract of Simarouba sp. can be used as a botanical insecticide effective in controlling P. xylostella by affecting its feeding, oviposition and embryonic development.”

 I think that the results presented in this study just indicate that there is deterrent activity of the Simarouba sp.  extracts. Therefore, it is too early to state that these extracts “can be used as a botanical insecticide...”. Further experiments are required to support this conclusion. Please improve.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The quality of English should be improved

Author Response

Comments and Suggestions for Authors

Round 1 – Reviewer 2

Manuscript ID: sustainability-3414907

Title: Evaluation of Bioinsecticides in the Control of Plutella xylostella (Linnaeus, 1758): A Laboratory Study for Large-Scale Implementation

We appreciate the reviewer's valuable suggestions. We inform that we understood the suggestions and some sentences have been altered as indicated by this reviewer and other reviewers. Language and textual fluency have also been improved. Scientific names were reviewed.

General comments

This MS describes the effect of aqueous extracts of Simarouba sp. leaves on oviposition, feeding and larval hatching of Plutella xylostella. This MS should be improved with concern to the English quality. In addition, the authors should determine and mention the Simarouba species. The title needs improvement to specify that some aqueous leaf extracts were studied and not some bioinsecticides. In addition, this MS would gain added value from the explicit determination of the chemical ingredients of the leaf extracts.

Response: We thank the reviewer for his comments and would like to inform him that MDPI English Language Editing Services has revised the English. Regarding the identification of the species, we sent it to several taxonomists and only obtained the genus as a true result. This is why we are presenting it in this format. The title has been improved, and the word "bioinsecticide" has been replaced by "botanical extract."

As for the chemical determination of the compounds, we based our choice for the chemical analysis on the availability of resources, such as suitable analytical techniques and sample processing resources. We recognize that identifying alkaloids and terpenes would be interesting once they are biologically active constituents, often associated with cytotoxicity and insecticidal properties. However, these analyses were not carried out due to specific instrumental or analytical limitations when the study was conducted. In addition, terpenes are not soluble in water, and this work seeks to better understand the effect of the aqueous extract (because it is widely used in vegetable gardens and by small and medium-sized producers). As this is a manuscript evaluating the insecticidal potential of Simarouba sp., we have focused on learning more about the plant's chemical composition, starting by identifying the presence of classes of compounds with insecticidal action. In future studies, we are working towards more comprehensive approaches to identifying and quantifying these compounds and using new solvents, such as hydroalcoholic extracts.

Comment 1: Abstract, lines 29-32. “The present study analyzed the effects of bioinsecticides of Simarouba sp. at concentrations of 0.1, 1, 5, and 10% and a control on the feeding preference, oviposition and embryonic development of P. xylostella.”

The authors studied the effect of aqueous extracts of Simarouba sp. Leaf and not of some bioinsecticides. Please improve.

Response:  Thank you for the comment. The requested alterations have been made.

Comment  2: Abstract, lines 32 to 33. The results showed that the bioinsecticides of Simarouba sp. decreased oviposition and feeding in P. xylostella, and a reduction in larval hatching was also observed, indicating ovicidal properties. Please improve with respect to grammar and syntax.

Response: We thank the reviewer for his comments.

Comments 3: Abstract, line 35. “Dietary concentrations of 10% and 5% caused…”. Please explain the meaning of dietary concentrations.

Response: Thank you for the observation. Unfortunately, it was a translation mistake, and we have already corrected it.

Comments 4: Abstract, lines 37-41.  “This study highlights the efficacy of bioinsecticides of Simarouba sp. in the control of the crucifer moth, as larval feeding and the number of individuals reaching the larval stage, the stage at which the insect causes losses to producers, were reduced. Thus, this laboratory study is expected to be successful if practiced on 40 a large scale.” Please improve with respect to grammar and syntax.

Response: We thank the reviewer for his comments and would like to inform him that MDPI English Language Editing Services has revised the English.

Comments 5: P. 97. The P. xylostella rearing methodology was adapted from Barros et al. [25]. The authors should briefly describe the rearing methodology.

Response: We inserted a brief description of the methodology in section (Line 107).

The P. xylostella stock was established from individuals collected from Brassica oleracea var. acephala ’s organic gardens in Itaporã, Mato Grosso do Sul, Brazil. The P. xylostella rearing methodology was adapted from Barros et al. [32].

The diamondback moth pupae were transferred to plastic cages (9 cm × 19 cm × 19 cm) until the moths emerged. The adults were fed with a solution of honey diluted in 10% distilled water. We used organic cabbage discs (Brassica oleracea var. acephala) and filter paper moistened with distilled water (measuring 9 cm Ø) as oviposition substrates. We transferred the oviposited discs to new plastic containers (30 cm × 15 cm × 12 cm). After the eggs hatched, P. xylostella larvae remained in these plastic containers until they reached the pupal stage. The larvae were fed organic cabbage leaves, which had been previously disinfected with 5% sodium hypochlorite. We sanitized the cages and replaced the cabbage leaves every 48 hours. Pupae were transferred back to the adult cage, and the cycle started again.

Comments 6: Line 108 to 109. After collection, the leaves were washed under running water, dried in a forced-air oven for 72 hours at 45 °C and grounded in an industrial knife mill until a fine powder was obtained. Drying the leaves at 45°C may cause substantial changes in the chemical composition. Please comment on it. In addition, give some information about the size of the leaf particles after grounding and clarify whether sieves were used.

Response: Thank you for the observation, we hadn’t noticed it in the text. The methodology followes by the laboratory uses 40ºC to dry leaves. The method can be observed in: https://doi.org/10.3390/plants11192656.

The dried leaves were ground in an industrial mill (model AC-035/81) designed to generate homogeneous powder. Although the exact particle size was not measured, studies with similar equipment suggest particles of approximately 0.84 mm, depending on the material and the mill setting. We did not use additional sieves after grinding, as the powder obtained had a uniform texture suitable for extraction.

Comments 7: Lines 112 to 115.  The authors mention that “ ..The maceration technique was used to obtain the bioinsecticides (extract aqueous) of Simarouba sp. (EA-S). Bioinsecticides with concentrations of 10, 5, 1 and 0.1% were obtained by adding 3 g of the powdered material to 30 ml of distilled water, 1.5 g to 30 ml, 0.3 g to 30 ml and 0.015 g to 30 ml. The powdered material was weighed on an analytical balance (Bel Mark Analytical Balance – 0.001 g). The solutions were left to stand for 24 hours in a refrigerator and were subsequently filtered using filter paper”.

These concentrations refer to the leaf powder and not to a bioinsecticide. Please improve.

Response: Thank you for your observation. We have changed the text according to the suggestions.

Comments 8: Line 130- 131. The authors mention that “...the leaf area consumed was measured using ImageJ software, and the food preference index (FPI) of Kogan and Goeden [26] was calculated...”  Please give a more detailed description of the method for determining food preference.

 Response: Thank you for the comment. We have changed the text according to the suggestions.

 The food preference index (FPI) of Kogan and Goeden [33] was calculated. The extract was classified as a phagostimulant if the index was greater than 1, neutral if it was equal to 1, and phagodeterrent if it was lower than 1. The formula FPI = 2A/(M+A) was used, where A is the consumed area of the treated discs and M is the consumed area of the untreated discs (Line 147).

Comments 9: Lines 141-142. The authors mention that ‘’’Subsequently, 4-cm Ø cabbage discs were immersed in EA-S at concentrations of 0.1, 1, 5 and 10%, and control discs were immersed in distilled water. Please explain the EA-S meaning. In addition, substitute “Subsequently” with “For the experiments…” Please improve.

Response: Thank you for the comment. The requested alterations have been made.

Comments 10: Lines 148-150. The authors mention that “…A moistened filter paper disc measuring 8 cm Ø and four cabbage discs measuring 4 cm Ø (two control discs (immersed in water) and two discs immersed in the EA-S) were added to each cage as oviposition substrates…. “ . If the filter paper is 8 cm in diameter and the leaf discs 4 cm, then the leaf discs are expected to attach each other. However, Fig.2, shows a distance between the leaf discs. Please clarify.

 Response: We thank the reviewer for pointing out the inconsistency in the description of the measurements. There was a mistake in the way the dimensions of the filter paper disks were described. The 24 cm Ø disks allowed adequate spacing between the disks to avoid overlapping, as shown in Figure 2.

Comments 11: Lines 177- 178. “...The experiment was conducted with a completely randomized design with 10 replicates and 3 subsamples…”. Please explain the meaning of sub-samples.

Response: Thank you for the observation. Our experiment design contained 30 samples (replicates). We corrected the text in the manuscript.

Comments 12: Line 179. The authors mention that the “…The data were subjected to analysis of variance (ANOVA) with a Gaussian distribution, and the means were compared using the t test at a 5% probability...”. Please explain which means were compared.

Response: Thank you for your observation. We compared the means of the consumed leaf area on each plate, which can be divided into two groups: control and treatment.  We've added a legend to Table 1 explaining that the means were compared by line.

*Means on the line followed by the same letter do not differ significantly according to the t test at a 5% probability level. ¹According to [26/34] (Line 285).

Comments 13: Lines 183 -185. The authors mention that”…The effect of the plant extract was evaluated using the FPI [26]. The extract was classified as a phagostimulant if the index was greater than 1, neutral if it was equal to 1, and 184 phagodeterrent if it was lower than 1, using the formula FPI = 2A/(M+A), where A is the 185 consumed area of the treated discs and M is the consumed area of the untreated discs”. This part does not present any statistical analysis and, therefore, should be transferred to the “Materials and Methods”. The same for the text in lines 197 to 202.

Response:  Thank you for the comment. The requested alterations have been made.

Comments 14: Line 212. The authors mention that “The extracts were lyophilized (Alpha 1–2 LD Plus) …” . They should add some information about the extracts.

 Response: Thank you for the comment. The requested alterations have been made.

 

2.7 Chemical composition

The aqueous extracts (AE-S), obtained according to the methods described in Section 2.2., were lyophilized using an Alpha 1–2LD Plus system under parameters of 0.045 mbar vacuum and a temperature of -42 °C. This process produced a 12% yield. The lyophilized aqueous extract was stored at 4 °C and protected from light until further testing. For analysis, the extract was then solubilized at a concentration of 1 mg/mL. Results were expressed as the average of four extracts prepared in this study, with each sample analyzed in triplicate.

Comments 15: In Table 2, it is shown that the number eggs laid in the control discs varies from 60.4 to146.0. The authors should discuss this high variability in egg production, of the control females, between the different treatments.

Response: The 10% concentration of AE-S caused an average reduction of 47.50 eggs/couple because the females died 8 days after the start of the test, while the males from the same treatment remained alive during the 10 days of evaluation. We believe that mortality among the females may have occurred because they came into direct contact with the 10% AE-S at the time of oviposition. Although they later preferred the control substrate for oviposition, this initial exposure to the extract during oviposition may have been enough to cause an adulticidal effect. We emphasize that couples had contact with control and extract at the same time because they were in the same cage (chance of choice - Figure 2).

Comments 16: In Table 3, the mean number (± SE) of hatched P. xylostella larvae from eggs exposed to different concentrations of Simarouba sp. aqueous extract is shown. However, the number of eggs tested is not shown and therefore, it is risky to make any comparisons. Please add information about the initial number of eggs tested and hatchability percentages.

Response: Thank you for the comment. We have inserted the initial number of eggs in table 3 (n=3).

Comments 17: The authors determined the quantities of tannins, phenolic compounds, and flavonoids of Simarouba sp. leaves extracts (Table 4). However, they have not determined the chemical compounds of the leaf extracts which could be of great importance.

Response: We removed table 4 and placed it in the text. We based our choice for the chemical analysis on the availability of resources, such as suitable analytical techniques and sample processing resources. We recognize that identifying alkaloids and terpenes would be interesting once they are biologically active constituents, often associated with cytotoxicity and insecticidal properties. However, these analyses were not carried out due to specific instrumental or analytical limitations when the study was conducted. In addition, terpenes are not soluble in water, and this work seeks to better understand the effect of the aqueous extract (because it is widely used in vegetable gardens and by small and medium-sized producers). As this is a manuscript evaluating the insecticidal potential of Simarouba sp., we have focused on learning more about the plant's chemical composition, starting by identifying the presence of classes of compounds with insecticidal action. In future studies, we are working towards more comprehensive approaches to identifying and quantifying these compounds and using new solvents, such as hydroalcoholic extracts.

Comments 18: Lines 344 -346. “Our results showed that the aqueous extract of Simarouba sp. can be used as a botanical insecticide effective in controlling P. xylostella by affecting its feeding, oviposition and embryonic development.”

 I think that the results presented in this study just indicate that there is deterrent activity of the Simarouba sp.  extracts. Therefore, it is too early to state that these extracts “can be used as a botanical insecticide...”. Further experiments are required to support this conclusion. Please improve.

Response: Thank you for your observation. We have made some alterations in our conclusion to describe the necessity of further tests, mainly on non-target organisms.

Our study identified that the aqueous extracts of Simarouba sp. compromised embryonic development, significantly reduced feeding and oviposition preference, and, at higher concentrations, ultimately prevented feeding by P. xylostella. Our initial laboratory studies demonstrated that this plant has great potential for use at a large-scale. However, further studies must be conducted to ensure its economic viability and toxicity against the environment and humans (Line 494).

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

A review of the manuscript: Evaluation of Bioinsecticides in the Control of Plutella xylostella (Linnaeus, 1758): A Laboratory Study for Large-Scale Implementation

To control the insect pests farmers extensively apply chemical insecticides which have negative effect on the environment and human health hazards. Therefore, it is important to switch to use of botanicals as well as plant extracts instead of chemical insecticides for the control of insect pest. They are cheaper, safe for environmental compatibility, exhibit low toxicity to non-target organisms. So results presented in reviewed manuscript is great of value.

The manuscript is overall well-written, well-structured and really clear. I think that the interpretations of the data to reach conclusions are fine. Discussion is interpreted in detail. I just have some comments presented below:

1.   Section 2.2. Bioinsecticides of Simarouba sp.:

-  it is worth to present a short description of Simarouba plant that was used as bioinsecticide, because there is not presented a specific species of this genus;

-  please provide the information how many leaves were used to the maceration technique to get needed quantity of powdered material. 

2.        Information on the number of replicates of each analysis contained in the section 2.6. Statistical analysis, should be transfer to sections 2.4., 2.5., respectively. 

3.        In the Discussion in the Oviposition subsection Authors started to use the abbreviation AE-S instead of EA-S for tested bioinsecticide. Is it literature data and refer to another substance, or is it an error made by Authors? Similarly information we can find in the further part of the Discussion and in Conclusions. This should be explain.

Author Response

Comments and Suggestions for Authors

Round 1 – Reviewer 3

Manuscript ID: sustainability-3414907

 Title: Evaluation of Bioinsecticides in the Control of Plutella xylostella (Linnaeus, 1758): A Laboratory Study for Large-Scale Implementation

 

We appreciate the reviewer's valuable suggestions. We inform that we understood the suggestions and some sentences have been altered as indicated by this reviewer and other reviewers. Language and textual fluency have also been improved. Scientific names were reviewed.

 Review: To control the insect pests farmers extensively apply chemical insecticides which have negative effect on the environment and human health hazards. Therefore, it is important to switch to use of botanicals as well as plant extracts instead of chemical insecticides for the control of insect pest. They are cheaper, safe for environmental compatibility, exhibit low toxicity to non-target organisms. So results presented in reviewed manuscript is great of value. The manuscript is overall well-written, well-structured and really clear. I think that the interpretations of the data to reach conclusions are fine. Discussion is interpreted in detail. I just have some comments presented below:

Section 2.2. Bioinsecticides of Simarouba sp.:

-  it is worth to present a short description of Simarouba plant that was used as bioinsecticide, because there is not presented a specific species of this genus;

 Authors: Thank you for your comment. We added a small description of the genus in the session.

Line 75: The Simaroubaceae family comprises 32 genera and more than 170 species [23]. In Brazil, this family presents six genera: Simaba and Simarouba, present throughout the country; Castela and Picrasma, located in the South; and Quassia and Picrolemma, predominant in the Amazon [24]. Simarouba is a medium-sized tree, reaching up to 20 meters in height, with a trunk that varies between 50 and 80 cm in diameter. It has shiny green leaves that are 20 to 50 cm long, small white flowers, and small red fruits [25,26].

Previous studies on Simarouba genus members have identified the presence of numerous bioactive compounds, such as alkaloids, quassinoids [23], triterpenes, and flavonoids [25], which can act as antifeedant substances [27,28] or interfere with the physiology of the insect pest [29-31]. In the central region of Mato Grosso do Sul, Brazil, (where this study was carried out), the Simarouba species were selected due to observations made on site, in which the absence of insect presence or attacks on the plant was noted.

 Review: please provide the information how many leaves were used to the maceration technique to get needed quantity of powdered material. 

 

Authors: Thank you for your comment.

Line 127: We washed the Simarouba sp. leaves (380 g) under running water, dried them in an oven (AC-035/81) with forced circulating air at 45 ± 3°C for 72 hours, pulverized them using a Willey knife mill, and sieved them through a 10 mm mesh (MA340/A). The resulting powder (86 g) was stored in plastic containers to protect it from moisture and light. We used part of the powdered material to obtain an aqueous extract (AE-S) from Simarouba sp., with concentrations of 10, 5, 1, and 0.1%, for use as a aqueous extract, as follows: (i) 3 g of the powder in 30 ml of distilled water, (ii) 1.5 g in 30 ml, (iii) 0.3 g in 30 ml, and (iv) 0.015 g in 30 ml, respectively. All AE-S were refrigerated for 24 hours and subsequently filtered using filter paper.

Review: Information on the number of replicates of each analysis contained in the section 2.6. Statistical analysis, should be transfer to sections 2.4., 2.5., respectively. 

Authors: Thank you for your comment. We have made a transference.

Review: In the Discussion in the Oviposition subsection Authors started to use the abbreviation AE-S instead of EA-S for tested bioinsecticide. Is it literature data and refer to another substance, or is it an error made by Authors? Similarly information we can find in the further part of the Discussion and in Conclusions. This should be explain.

Authors: Thank you for your commentary and for your precise analysis. It was a translation mistake, and we have already correct it.

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The subject merits study and the authors make a contribution the field of research. However, before it can be published, I suggest that several changes be made. 

The introduction makes some assumptions that are either generalizations that are not fully supported by the literature or need to be qualified. Specifically, botanical insecticides are not always non-toxic. The authors do not adequately explain the mode of action of Simarouba spp. in the introduction and leave it to the discussion to speculate on possible hypotheses that can be tested. The authors should explain why they selected this random genus to test on the target pest and not, say, Quassia spp. or conduct an experiment that included several candidate botanicals. 

Botanicals have multiple modes of action and varying potency in active ingredients. The quality control needs further explanation.

The results of the control are not consistently reported.

The authors should do a better job of explaining why some constituents were analyze and others were not. Alkaloids are mentioned in the text but I don't see separate analytical results for them. The authors note their importance as antifeedants. Terpenes / terpenoids are often cytotoxic constituents in botanical insecticides and these are not even mentioned, and no analytical results for them are reported. At least acknowledge them as biologically active constituents and explain why they were not analyzed.

I suggest that before conducting field experiments, the authors perform toxicity tests on beneficial insects and non-target species such as Apis mellifera. 

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The authors rely too much on a passive voice. While I understand that some scientists prefer the passive voice, I don't. For the most part, a reader can understand what is going on, but especially in the Materials and Methods section, the passive voice and omitted details lead to ambiguity and confusion. Who is doing what and why?

Author Response

Comments and Suggestions for Authors

Round 1 – Reviewer 4

Manuscript ID: sustainability-3414907

 

Title: Evaluation of Bioinsecticides in the Control of Plutella xylostella (Linnaeus, 1758): A Laboratory Study for Large-Scale Implementation

 

We appreciate the reviewer's valuable suggestions. We inform that we understood the suggestions and some sentences have been altered as indicated by this reviewer and other reviewers. Language and textual fluency have also been improved. Scientific names were reviewed.

 

Reviewer: The subject merits study and the authors make a contribution the field of research. However, before it can be published, I suggest that several changes be made.

The introduction makes some assumptions that are either generalizations that are not fully supported by the literature or need to be qualified. Specifically, botanical insecticides are not always non-toxic. The authors do not adequately explain the mode of action of Simarouba spp. in the introduction and leave it to the discussion to speculate on possible hypotheses that can be tested. The authors should explain why they selected this random genus to test on the target pest and not, say, Quassia spp. or conduct an experiment that included several candidate botanicals.

Authors: Thank you for your commentary. We recognize that botanical insecticides are not always non-toxic, and we have reformulated the text to reflect this based on the literature. We would like to clarify that we based our choice of Simarouba sp. for this study on on-site observations, where, on all visits, we noted the absence of insects or damage caused by them on the plant. On the other hand, insects such as leaf-cutting ants and lepidopteran pests were present on the surrounding plants. We included this information in the introduction to justify the choice of plant (Line 84). We chose not to include other species, such as Quassia spp., in this initial study in order to concentrate efforts on a single plant with high potential, ensuring greater depth in investigations such as this manuscript and new tests that will be carried out, such as the effect on non-target organisms. We also have changed the possible mechanisms of action in the introduction as suggested (Line 81).

 

Reviewer: Botanicals have multiple modes of action and varying potency in active ingredients. The quality control needs further explanation.

Authors: We appreciate the reviewer's comment. As botanical insecticides contain a complex mixture of chemical compounds with multiple potential modes of action, some compounds may act synergistically, while others have independent functions. In this sense, quality control must identify the main active ingredients or chemical markers contributing significantly to the desired biological effect. We would like to point out that we have not yet reached this research stage, but we hope to soon be able to provide clearer information on studies that the research group is currently carrying out. However, we recognize the importance of good quality control and we try to follow a few steps: 1) standardization of collection, such as location, time, healthy and fully expanded leaves; 2) standardization of the extraction process, including drying, grinding and aqueous extraction methodology, to ensure repeatability; 3) chemical characterization of key compound classes (phenolic compounds, tannins and flavonoids) using spectrophotometric methods to monitor potency and consistency; and 4) bioassays to verify biological activity, at different stages of insect-pest development and evaluating the extract in different ways (antibiosis and antixenosis).

 

Reviewer: The results of the control are not consistently reported.

Authors: We thank the reviewer for highlighting the inconsistency in reporting the control results. We have carefully reviewed the text to ensure the control values are presented clearly and consistently throughout the manuscript. Besides that, we have found a new way to explain feeding and oviposition results (Figure 4 and 6). Regarding tables 1 and 2, the control can be observed next to the column “extract”. Preference index and its classification were accessed by the comparison between control and extract per concentration. Each table line represents an extract concentration and contains the parameter (consumed leaf area/number of eggs for both extract and control), calculated preference index, and its meaning.

Once each cage/ plate contained both extract and control, there is a control for each treatment, leading to this data organization. Each concentration has a different control because oviposition and food preference tests were carried out with the extract and the control inserted in the same cage or Petri dish. This allows us to evaluate the choice of moths and caterpillars between the two substrates. It’s possible to better understand the design of the experiments in Figures 1 and 2.

The change requested in table 3 has been made.

 

Reviewer: The authors should do a better job of explaining why some constituents were analyze and others were not. Alkaloids are mentioned in the text but I don't see separate analytical results for them. The authors note their importance as antifeedants. Terpenes / terpenoids are often cytotoxic constituents in botanical insecticides and these are not even mentioned, and no analytical results for them are reported. At least acknowledge them as biologically active constituents and explain why they were not analyzed.

Authors: We based our choice on analyzing specific chemical groups on the availability of resources, such as suitable analytical techniques and sample processing resources. Although alkaloids have been mentioned and their relevance as antifeedants recognized, their detailed analysis was limited by the availability of specific methods within the scope of this work. However, their presence was addressed in a general way to contextualize their importance and potential contribution to the effects observed. We recognize terpenes are biologically active constituents, often associated with cytotoxicity and insecticidal properties. Despite this, their analysis was not carried out due to specific instrumental or analytical limitations at the time that time. In addition, terpenes are not soluble in water, and the present work seeks to better understand the effect of the aqueous extract (because it is widely used in vegetable gardens and by small and medium-sized producers). The focus was on the constituents that were most directly relevant to the specific objectives of the work. In future studies, we intend to explore more approaches to identify and quantify these compounds and the use of hydroalcoholic extracts. We recognize the biological importance of unanalyzed constituents. Although they were not the subject of analysis in this study, we acknowledge the relevance of alkaloids, terpenes, and other biologically active constituents in the context of the effects observed. This gap is considered a limitation of the work and will be addressed in future publications or through complementary studies that can explore these compounds in greater detail.

 

Reviewer: I suggest that before conducting field experiments, the authors perform toxicity tests on beneficial insects and non-target species such as Apis mellifera. 

Authors: Thank you for your comment, the authors agree with your perspective. We are paying close attention to the tests mentioned above, and we have already begun tests with non-target organisms as a new stage of this study, using Apis mellifera, Melipona quadrifasciata and the endoparasitoid Tetrastichus howardi. The model organism used as a bioindicator will be Caenorhabditis elegans. However, we consider that field testing is an important next step, as the effectiveness of the extracts needs to be confirmed in the field. In the conclusion, we point out the need for further studies, especially on non-target organisms.

 

Reviewer: (line 61) Not always true. Insects resistant to pyrethrum were first noted in the literature in the 1970s.

(line 63) Again, not always true, and depends on whether the whole plant or an extract is applied. Rotenone is highly toxic to fish. Strychnine from Nux vomica is highly toxic to most vertebrates. So is nicotine extracted from tobacco.

Authors: Thank you for your suggestion. The text has been changed to reinforce that there are exceptions.

 

Reviewer: (line 65) Better to say that botanicals have multiple modes of action and do not always act as biocides.

Authors: Thank you for your suggestion. The text has been changed.

 

Reviewer: (line 89) General comment: The authors write much of this section in the passive voice—or to put it in the passive voice—the passive voice is used in much of this section. Consider using the active voice, particularly in parts where the passive voice causes ambiguity of meaning.

Authors: Thank you for your suggestion, we changed the entire Section 2 (Materials and Methods) to the active voice.

 

Reviewer: (line 101) By whom? The authors or an authorized third party?

Authors: The text has been changed in order to clarify its meaning.

 

Reviewer: (line 184) All of these subsections are written in the passive voice. I'm calling out this one because FPI needs more explanation and it appears to involve some subjective judgment of the experimenter. In any case, the reader deserves a short summary of the FPI methodology without needing to pull up the cited source.

Authors: A brief commentary about the formula used to calculate FPI was added to the section.

 

Reviewer: (line 420) Were alkaloid concentrations analyzed separately? If so, please report the results.

Authors: Alkaloids and quassinoids were not tested separately.

 

Reviewer: (line 421) Are you planning to test that hypothesis?

Authors: Future studies will test this hypothesis by evaluating the specific impact of each chemical constituent on P. xylostella, as well as their potential effects on non-target organisms, including beneficial arthropods, like Apis mellifera, Melipona quadrifasciata, parasitoid Tetrastichus howardi and the nematode Caenorhabditis elegans.

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

I am pleased that the authors have accepted most of my suggestions. Obviously some of my recommendations would require further research and I encourage them to follow up if possible. 

Comments on the Quality of English Language

The article has several places where the text got garbled in the revision process when the original text was not deleted. On January 31, I submitted more detailed information on the lines to edit. I don't have time to do it all over again. 

Author Response

Comments and Suggestions for Authors

Round 2 – Reviewer 4

Manuscript ID: sustainability-3414907

 

Title: Evaluation of Bioinsecticides in the Control of Plutella xylostella (Linnaeus, 1758): A Laboratory Study for Large-Scale Implementation

 

Thank you for your important observations during rounds one and two. We followed orientations from round one, and hoping to make it easier to understand, we decided to send you our manuscript without the alteration markers. We described all the lines where we have been making alterations in this letter. We also adjusted tables and images organization. Also, we would like to inform you that we submitted our manuscript to MDPI English Language Editing Services for revision.

Reviewer: The subject merits study and the authors make a contribution the field of research. However, before it can be published, I suggest that several changes be made.

The introduction makes some assumptions that are either generalizations that are not fully supported by the literature or need to be qualified. Specifically, botanical insecticides are not always non-toxic. The authors do not adequately explain the mode of action of Simarouba spp. in the introduction and leave it to the discussion to speculate on possible hypotheses that can be tested. The authors should explain why they selected this random genus to test on the target pest and not, say, Quassia spp. or conduct an experiment that included several candidate botanicals.

Authors: Thank you for your commentary. We recognize that botanical insecticides are not always non-toxic, and we have reformulated the text to reflect this based on the literature. We would like to clarify that we based our choice of Simarouba sp. for this study on on-site observations, where, on all visits, we noted the absence of insects or damage caused by them on the plant. On the other hand, insects such as leaf-cutting ants and lepidopteran pests were present on the surrounding plants. We chose not to include other species, such as Quassia spp., in this initial study in order to concentrate efforts on a single plant with high potential, ensuring greater depth in investigations such as this manuscript and new tests that will be carried out, such as the effect on non-target organisms. We have added the information requested in the introduction, line 75-86:

“The Simaroubaceae family comprises 32 genera and more than 170 species [23]. In Brazil, this family presents six genera: Simaba and Simarouba, present throughout the country; Castela and Picrasma, located in the South; and Quassia and Picrolemma, predominant in the Amazon [24]. Simarouba is a medium-sized tree, reaching up to 20 meters in height, with a trunk that varies between 50 and 80 cm in diameter. It has shiny green leaves that are 20 to 50 cm long, small white flowers, and small red fruits [25,26]. Previous studies on Simarouba genus members have identified the presence of numerous bioactive compounds, such as alkaloids, quassinoids [23], triterpenes, and flavonoids [25], which can act as antifeedant substances [27,28] or interfere with the physiology of the insect pest [29-31]. In the central region of Mato Grosso do Sul, Brazil, (where this study was carried out), the Simarouba species were selected due to observations made on site, in which the absence of insect presence or attacks on the plant was noted”.

 

Reviewer: Botanicals have multiple modes of action and varying potency in active ingredients. The quality control needs further explanation.

Authors: We appreciate the reviewer's comment. As botanical insecticides contain a complex mixture of chemical compounds with multiple potential modes of action, some compounds may act synergistically, while others have independent functions. In this sense, quality control must identify the main active ingredients or chemical markers contributing significantly to the desired biological effect. We would like to point out that we have not yet reached this research stage, but we hope to soon be able to provide clearer information on studies that the research group is currently carrying out. However, we recognize the importance of good quality control and we try to follow a few steps: 1) standardization of collection, such as location, time, healthy and fully expanded leaves; 2) standardization of the extraction process, including drying, grinding and aqueous extraction methodology, to ensure repeatability; 3) chemical characterization of key compound classes (phenolic compounds, tannins and flavonoids) using spectrophotometric methods to monitor potency and consistency; and 4) bioassays to verify biological activity, at different stages of insect-pest development and evaluating the extract in different ways (antibiosis and antixenosis).

 

Reviewer: The results of the control are not consistently reported.

Authors: We thank the reviewer for highlighting the inconsistency in reporting the control results. We have carefully reviewed the text to ensure the control values are presented clearly and consistently throughout the manuscript. Besides that, we have found a new way to explain feeding (Figure 4 – line 276) and oviposition results (Figure 6 – line 317).

 

 

Figure 4. Leaf area consumed (± SE) and food preference index of Plutella xylostella exposed to cabbage discs treated with bioinsecticides of Simarouba sp. at different concentrations. All grouped bars (Control versus Concentration) differ significantly according to the t-test at a 5% probability level. The preference index was calculated at 0.11, 0.01, 0.00, and 0.00 to the concentrations of 0.01, 1, 5 and 10%, respectively. Thus, all concentrations were classified as Phagodeterrent, according to Kogan and Goeden [33/36].

Figure 6. Number of eggs (± SE) and oviposition preference index of Plutella xylostella on cabbage discs treated with bioinsecticides of Simarouba sp. at different concentrations. Grouped bars (Control versus Extract) are significantly different when compared by the F test (P <0.05) from the generalized linear model with a Poisson distribution with overdispersion (quasiPoisson model) at a 5% probability. The preference index was calculated at 0.60, 0.52, 0.55, and 0.60 to the concentrations of 0.01, 1, 5 and 10%, respectively. Thus, all concentrations were classified as Ovideterrent, according to Kogan and Goeden [33/36].

 

Regarding tables 1 and 2 (Line 287 and 333), the control can be observed next to the column “extract”. Preference index and its classification were accessed by the comparison between control and extract per concentration. Each table line represents an extract concentration and contains the parameter (consumed leaf area/number of eggs for both extract and control), calculated preference index, and its meaning.

Once each cage/ plate contained both extract and control, there is a control for each treatment, leading to this data organization. Each concentration has a different control because oviposition and food preference tests were carried out with the extract and the control inserted in the same cage or Petri dish. This allows us to evaluate the choice of moths and larvae between the two substrates. It’s possible to better understand the design of the experiments in Figures 1 and 2 (Line 160 and 213).

 

The change requested in table 3 has been made.

Table 3. Mean number (± SE) of hatched P. xylostella larvae from eggs exposed to different Simarouba sp. aqueous extract concentrations.

Concentrations of Simarouba sp. aqueous extract
	AE-S 10%	AE-S 5%	AE-S 1%	AE-S 0.1%	Control
Number of hatched larvae	7.63 ± 0.74 b n= 10	7.92 ± 0.51 b n= 10	7.15 ± 0.41 b n= 10	8.67 ± 0.33 ab n= 10	9.99 ± 0.00 a n= 10
F and P value	F= 8.94; P< 0.0001

*Means were compared by the F test (P <0.05) from the generalized linear model with a binomial distribution with overdispersion (quasibinomial) at a 5% probability.

 

Reviewer: The authors should do a better job of explaining why some constituents were analyze and others were not. Alkaloids are mentioned in the text but I don't see separate analytical results for them. The authors note their importance as antifeedants. Terpenes / terpenoids are often cytotoxic constituents in botanical insecticides and these are not even mentioned, and no analytical results for them are reported. At least acknowledge them as biologically active constituents and explain why they were not analyzed.

Authors: We based our choice on analyzing specific chemical groups on the availability of resources, such as suitable analytical techniques and sample processing resources. Although alkaloids have been mentioned and their relevance as antifeedants recognized, their detailed analysis was limited by the availability of specific methods within the scope of this work. However, their presence was addressed in a general way to contextualize their importance and potential contribution to the effects observed. We recognize terpenes are biologically active constituents, often associated with cytotoxicity and insecticidal properties. Despite this, their analysis was not carried out due to specific instrumental or analytical limitations at the time that time. In addition, terpenes are not soluble in water, and the present work seeks to better understand the effect of the aqueous extract (because it is widely used in vegetable gardens and by small and medium-sized producers). The focus was on the constituents that were most directly relevant to the specific objectives of the work. In future studies, we intend to explore more approaches to identify and quantify these compounds and the use of hydroalcoholic extracts. We recognize the biological importance of unanalyzed constituents. Although they were not the subject of analysis in this study, we acknowledge the relevance of alkaloids, terpenes, and other biologically active constituents in the context of the effects observed. This gap is considered a limitation of the work and will be addressed in future publications or through complementary studies that can explore these compounds in greater detail.

 

Reviewer: I suggest that before conducting field experiments, the authors perform toxicity tests on beneficial insects and non-target species such as Apis mellifera. 

Authors: Thank you for your comment, the authors agree with your perspective. We are paying close attention to the tests mentioned above, and we have already begun tests with non-target organisms as a new stage of this study, using Apis mellifera, Melipona quadrifasciata and the endoparasitoid Tetrastichus howardi. The model organism used as a bioindicator will be Caenorhabditis elegans. However, we consider that field testing is an important next step, as the effectiveness of the extracts needs to be confirmed in the field. In the conclusion (Line 496), we point out the need for further studies, especially on non-target organisms.

“Our study identified that the aqueous extracts of Simarouba sp. compromised embryonic development, significantly reduced feeding and oviposition preference, and, at higher concentrations, ultimately prevented feeding by P. xylostella. Our initial laboratory studies demonstrated that this plant has great potential for use at a large-scale. However, further studies must be conducted to ensure its economic viability and toxicity against the environment and humans”.

 

Reviewer: (line 61) Not always true. Insects resistant to pyrethrum were first noted in the literature in the 1970s.

(line 63) Again, not always true, and depends on whether the whole plant or an extract is applied. Rotenone is highly toxic to fish. Strychnine from Nux vomica is highly toxic to most vertebrates. So is nicotine extracted from tobacco.

Authors: Thank you for your suggestion. We agree with the reviewer and the text has been changed to reinforce that there are exceptions (Line 60-64).

“Botanical insecticides have reemerged as an alternative to chemical control [17] since, generally, they do not favor the evolution of resistance in herbivorous insects [18,19], and are less likely to be toxic to soil, water, and nontarget organisms such as natural predators, pollinators, vertebrates [20], and invertebrates [21]. In addition, they are both low-cost and easy to acquire, apply, and manage [22]”.

 

Reviewer: (line 65) Better to say that botanicals have multiple modes of action and do not always act as biocides.

Authors: Thank you for your suggestion. The text has been changed (Line 65).

“Botanical insecticides have multiple modes of action and do not always act as biocides [21]. These specific characteristics of plant-based insecticides are essential for integrated pest management in that, even if they do not cause the immediate death of insects, they can exert sublethal effects, such as phagodeterrence, ovideterrence [23,24], reduction in the weight of immature insects, reduction in fertility and fecundity and changes in insect development [21], significantly reducing pest populations and decreasing the damage caused to crops”.

 

Reviewer: (line 89) General comment: The authors write much of this section in the passive voice—or to put it in the passive voice—the passive voice is used in much of this section. Consider using the active voice, particularly in parts where the passive voice causes ambiguity of meaning.

Authors: Thank you for your suggestion, we changed the entire Section 2 (Materials and Methods) to the active voice (From line 100). Here's an example of the changes we've made (Line 110-119):

“The diamondback moth pupae were transferred to plastic cages (9 cm × 19 cm × 19 cm) until the moths emerged. The adults were fed with a solution of honey diluted in 10% distilled water. We used organic cabbage discs (Brassica oleracea var. acephala) and filter paper moistened with distilled water (measuring 9 cm Ø) as oviposition substrates. We transferred the oviposited discs to new plastic containers (30 cm × 15 cm × 12 cm). After the eggs hatched, P. xylostella larvae remained in these plastic containers until they reached the pupal stage. The larvae were fed organic cabbage leaves, which had been previously disinfected with 5% sodium hypochlorite. We sanitized the cages and replaced the cabbage leaves every 48 hours. Pupae were transferred back to the adult cage, and the cycle started again”.

 

Reviewer: (line 101) By whom? The authors or an authorized third party?

Authors: The text has been changed in order to clarify its meaning (Line 122):

“We collected leaves of fully expanded Simarouba sp. from an area dominated by Cerrado in Campo Grande, Mato Grosso do Sul, Brazil (latitude: 21°13’28” S, longitude: 54°11’28” W; altitude: 437 m)”.

 

Reviewer: (line 184) All of these subsections are written in the passive voice. I'm calling out this one because FPI needs more explanation and it appears to involve some subjective judgment of the experimenter. In any case, the reader deserves a short summary of the FPI methodology without needing to pull up the cited source.

Authors: Thank you for your suggestion. We have inserted the active voice in the methodology. A brief commentary about the formula used to calculate FPI was added (Line 147-154).

“After 24 hours, the cabbage discs were removed and scanned (Figure 1), the leaf area consumed was measured using ImageJ software, and the food preference index (FPI) of Kogan and Goeden was calculated [33]. We calculated FPI through the consumed leaf area using the following formula: FPI = 2A/(M+A). A is the consumed area of the treated discs, and M is the consumed area of the untreated discs. We obtained an index between 0 and 2. The extract was classified as a phagostimulant, neutral or phagodeterrent. Phagostimulant if the index was greater than 1, neutral if it was equal to 1, and phagodeterrent if it was lower than 1”.

 

Reviewer: (line 420) Were alkaloid concentrations analyzed separately? If so, please report the results.

Authors: Alkaloids and quassinoids were not tested separately.

 

Reviewer: (line 421) Are you planning to test that hypothesis?

Authors: Future studies will test this hypothesis by evaluating the specific impact of each chemical constituent on P. xylostella, as well as their potential effects on non-target organisms, including beneficial arthropods, like Apis mellifera, Melipona quadrifasciata, parasitoid Tetrastichus howardi and the nematode Caenorhabditis elegans.

Reviewer: (line 447) Before doing so field experiments, I would like to see more information about the acute toxicity to humans…

Thank you for your comment, the authors agree with your perspective. We are paying close attention to the tests mentioned above, and we have already begun tests with non-target organisms as a new stage of this study, using Apis mellifera, Melipona quadrifasciata and the endoparasitoid Tetrastichus howardi. The model organism used as a bioindicator will be Caenorhabditis elegans. However, we consider that field testing is an important next step, as the effectiveness of the extracts needs to be confirmed in the field. In the conclusion, we point out the need for further studies, especially on non-target organisms (Line 496).

“Our study identified that the aqueous extracts of Simarouba sp. compromised embryonic development, significantly reduced feeding and oviposition preference, and, at higher concentrations, ultimately prevented feeding by P. xylostella. Our initial laboratory studies demonstrated that this plant has great potential for use at a large-scale. However, further studies must be conducted to ensure its economic viability and toxicity against the environment and humans”.

Author Response File: Author Response.pdf